This application is for an individual fellowship for an MD-PhD student, with a research training plan designed to aid her long-term goal of becoming an independent physician-scientist. The accurate segregation of chromosomes during mitosis is essential for the survival and development of all eukaryotes. Defects in chromosome segregation causes cell death or aneuploidy, which causes congenital defects or spontaneous abortions in embryogenesis, and contributes to cancer progression in somatic cells. Accurate chromosome segregation requires attachment of mitotic spindle microtubules to the kinetochore, which is assembled on the chromosomal centromere. Centromere location is specified epigenetically by the histone H3 variant termed centromere protein A (CENP-A), which directly binds to another essential centromere protein CENP-C. Preliminary studies using the method of hydrogen-deuterium exchange coupled to mass spectrometry (H/DX-MS) suggest that CENP-C binding alters structural properties of CENP-A nucleosomes, suggesting that CENP-C helps define the architecture of functional centromeric chromatin. In Aim 1, H/DX-MS studies will be extended to test whether such structural alterations are specific to CENP-A nucleosomes, and CENP-C mutants will be tested in cells to investigate whether abolishment of CENP-C-induced alterations is linked to centromere defects. The second focus of this proposal is the mechanisms ensuring accurate propagation of centromere location across cell cycles. Cyclin- dependent kinase (Cdk) 1/2 regulates the timing for the loading of newly expressed CENP-A molecules into newly replicated centromeric DNA, possibly by phosphorylating key player(s) in the CENP-A-loading pathway. In Aim 2, the method of stable isotope labeling by amino acids in cell culture (SILAC) will be used to identify Cdk1/2-dependent phosphorylation sites on affinity- purified CENP-A-containing complexes, and a fluorescent pulse-chase labeling approach will be used in cells to identify the phosphorylation sites that control the timing of CENP-A loading. These experiments will shed key insight into how centromere location is accurately specified and propagated over time, which are crucial processes for maintaining genomic stability.